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Short communication
Isolation and characterization of phosphate solubilizing bacteria
from the rhizosphere of crop plants of Korea
Heekyung Chunga, Myoungsu Parka, Munusamy Madhaiyana, Sundaram Seshadria,
Jaekyeong Songb, Hyunsuk Chob, Tongmin Saa,*
aDepartment of Agricultural Chemistry, Chungbuk National University, 48, Gaeshing Dong,
Heungduk Gu, Cheongju, Chungbuk 361-763, South KoreabKorean Agricultural Culture Collection (KACC), National Institute of Agricultural Biotechnology, Suwon 441-707, South Korea
Received 6 April 2004; received in revised form 22 November 2004; accepted 21 February 2005
Abstract
Whole-cell fatty acids methyl ester (FAME) profile and 16S rDNA sequence analysis were employed to isolate and identify the bacterial
groups that actively solubilized phosphates in vitro from rhizosphere soil of various crops of Korea. Out of several hundred colonies that
grew on Pikovskaya’s medium 13 best isolates were selected based on the solubilization of insoluble phosphates in liquid culture and further
characterized and identified. They were clustered under the genera Enterobacter, Pantoea and Klebsiella and the sequences of three
representative strains were deposited in the GenBank nucleotide sequence data library under the accession numbers AY335552, AY335553,
AY335554.
q 2005 Published by Elsevier Ltd.
Keywords: Phosphate solubilization; Pantoea agglomerans; Enterobacter aerogenes; Klebsiella sp.
Microorganisms capable of producing a halo/clear zone
due to solubilization of organic acids in the surrounding
medium (Singal et al., 1991) are selected as potential
phosphate solubilizers (Das, 1989) and are routinely
screened in the laboratory by a plate assay method
(Gerretson, 1948) using either Pikovskaya agar (Pikovskaya,
1948) or Sperber agar (Sperber, 1958). Several reports on
bacteria and fungi isolated from soil have evaluated their
mineral phosphate solubilizing (MPS) activity with various P
sources such as calcium phosphate tribasic [Ca3(PO4)2]
(Illmer and Schinner, 1995), iron phosphate (FePO4) (Jones
et al., 1991) and aluminium phosphate (AlPO4) (Illmer et al.,
1995). An increase in P availability to plants through the
inoculation of PSBs has also been reported previously in pot
experiments and under field conditions (Banik and Dey,
1981; Chabot et al., 1996; deFreitas et al., 1997; Zaidi et al.,
2003).
0038-0717/$ - see front matter q 2005 Published by Elsevier Ltd.
doi:10.1016/j.soilbio.2005.02.025
* Corresponding author. Tel.: C82 43 261 2561; fax: C82 43 271 5921.
E-mail address: [email protected] (T. Sa).
Since the knowledge on the diversity of phosphate
solubilizing bacteria (PSB) in Korean soils is lagging, an
attempt to isolate and identify PSB through biochemical and
molecular methods was made. The rhizosphere soil samples
collected and transferred under aseptic conditions were
stored in an ice pack at 4 8C in the laboratory. One milliliter
of the appropriate (10K5–10K7) dilutions of the soil samples
was plated on Pikovskaya’s medium (Pikovskaya, 1948) for
the isolation of PSB. The colonies distinguished by
producing halo zones, were identified and sub-cultured
(Table 1). As the plate assay is not considered a reliable
method in determining a strain as phosphate solubilizer
(Johri et al., 1999), the pure cultures were further screened
in liquid medium containing Ca3(PO4)2, AlPO4 and FePO4
at a concentration of 5 g LK1 as insoluble P sources. The
cultures supernatant obtained by centrifugation was passed
through a 0.45 mM Millipore filter (Sartorius) and the
inorganic phosphate content of the culture filtrate was
determined by the molybdenum blue method (Murphy and
Riley, 1962). Autoclaved medium served as a control for
each set. All the isolates solubilized Ca3(PO4)2 to a greater
extent than AlPO4 and FePO4 with AlPO4 exhibiting poor
solubilization (Table 2). Even the isolates that did not
Soil Biology & Biochemistry 37 (2005) 1970–1974
www.elsevier.com/locate/soilbio
Table 1
Location, soil series, crops and colony morphology of PSB isolates
Location Soil series Crops (scientific name) Isolates Colony morphology
Gae Sin Dong Yesan Spring onion (Allium fistulosum L.) HK 11-1 White, slender
Yesan Pepper (Capsicum annuum L.) HK 14-1 White, circular
Yesan Spring onion HK 17-1 White, circular
Yesan Sesame (Sesamum indicum L.) HK 18-3 White, circular
Gang Seo Dong Sangju Sesame HK 20-1 White, circular
Sangju Pepper HK 23-2 White, circular
Sangju Pepper HK 24 White, circular
Hyeong Dong Ri Sangju Spring onion HK 34-1 White, circular
Sangju Spring onion HK 34-2 White, circular
Sek Pan Ri Sachon Rice (Oryza sativa L.) HK 52-1 White, circular
Jeung Pyung Sachon Rice HK 68-1 Yellow, circular
Sachon Rice HK 68-3 White, circular
Bong Yang Up Sachon Rice HK 69 White, circular
H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974 1971
perform well in plate assays exhibited significant phos-
phates solubilization in the liquid cultures. These isolates
presumably identified as PSBs further characterized by a
series of biochemical reactions as per the Bergey’s Manual
of Systemic Bacteriology (Holt et al., 1994) were Gram-
negative rods with positive for catalase activity and negative
for oxidase activity, H2S production, gelatin, starch and
lipid hydrolysis (Table 3).
The isolates were identified based on whole-cell cellular
fatty acids, derivatized to methyl esters, i.e. FAMEs and
analyzed by gas chromatography (GC) using the MIDI
system (MIDI, Newark, DE). The analysis was performed
using the Sherlock Microbial Identification system TSBA
4.0 software and library general system software version
4.1. Qualitative and quantitative differences in the fatty acid
profiles were used to compute the distance for each strain
relative to the strains in the library (Sasser, 1990a,b; Sasser
and Wichman, 1991). Genomic DNA was extracted by the
phenol/chloroform method (Sambrook et al., 1989) and
amplified using PCR amplification of the 16S ribosomal
Table 2
Solubilization of inorganic phosphates by the PSB isolates in liquid cultures
Isolates Liquid culture (mg P mLK1)
Ca3(PO4)2 A
HK 11-1 96.2G5.9
HK 14-1 127.2G2.1 1
HK 17-1 113.7G3.3
HK 18-3 121.7G2.7
HK 20-1 142.1G2.1
HK 23-2 114.3G8.4
HK 24 136.4G1.1
HK 34-1 138.6G5.7
HK 34-2 126.9G2.2 1
HK 52-1 107.7G2.1
HK 68-1 119.2G5.3
HK 68-3 107.5G2.2
HK 69 113.0G5.7
Liquid cultures were assayed after seven days. Ca3(PO4)2, tricalcium phosphate;
DNA (16S rDNA). fD1 (5 0-AGAGTTTGATCCTGGCT-
CAG-3 0) and rP2 (3 0-ACGGCTACCTTGTTACGACTT-5 0)
primers (Weisburg et al., 1991) were used. A GeneAmp
PCR System (Perkin–Elmer Co., Norwalk, CT) with Taq
DNA polymerase (Promega Co., Southampton, England)
was used for PCR (Park et al., in press). The sequencing was
performed using Big-Dye Terminator Cycle Sequencing
and an ABI Prism 310 Genetic Analyzer (Tokyo, Japan).
The phylogenetic tree for the data sets was inferred by the
neighbor-joining method using the neighbor-joining pro-
gram, MEGA version 2.0 (Kumar et al., 1993).
The GC-FAME analysis placed most of the isolates
under Enterobacter sp., Klebsiella sp., and Pantoea sp. that
are grouped under one family, Enterobacteriaceae. Results
of 16S rDNA identification agreed with that of GC-FAME
for five PSBs at the genus level in Enterobacter and
Klebsialla (Table 4). Isolation of bacteria belonging to the
family Enterobacteriaceae from various soils, and their MPS
activities has also been reported earlier (Kim et al., 1997,
1998; Vassilev et al., 1997, 1999; Remus et al., 2000).
lPO4 FePO4
8.0G5.0 18.8G6.3
3.8G1.3 22.9G1.7
4.3G0.9 4.5G0.9
3.7G0.7 10.2G1.1
5.1G1.6 24.4G1.1
9.9G0.9 18.8G4.3
5.7G0.6 19.3G1.0
5.9G1.1 27.2G3.7
0.7G1.3 12.6G1.0
6.3G0.4 15.5G6.4
8.7G0.4 20.7G4.6
7.2G0.6 49.1G2.2
4.1G1.7 49.5G4.7
AlPO4, aluminium phosphate; FePO4, ferric phosphate.
Table 3
Biochemical characteristics of the PSB isolates
Biochemical reactions PSB isolates
HK
11-1
HK
14-1
HK
17-1
HK
18-3
HK
20-1
HK
23-2
HK
24
HK
34-1
HK
34-2
HK
52-1
HK
68-1
HK
68-3
HK
69
Gram staining K K K K K K K K K K K K KCatalase C C C C C C C C C C C C C
Oxidase K K K K K K K K K K K K K
IMViC test
Indole production K K K K K K K K K K K K K
Methyl red K K K K K K K K K K K K K
Voges-Proskauer C C C C C C C C C C C C C
Citrate (Simmons) C C C C C C C C C C C C CLysine decarboxylase K K K C K C K K K K K C C
Arginine dihydrolase C C C C C K C C K C C K K
Ornithine decarboxylase C C C C C K K C K C C K K
Carbon source utilization
Sucrose C C C C C C C C C C C C C
Fructose C C C C C C C C C C C C C
Glucose C C C C C C C C C C C C C
Glycerol C C C C C C C C C C C C CMaltose C C C C C C C C C C C C C
Mannitol C C C C C C C C C C C C C
Inositol C C C C C C C C C C C C CDulicitol K K K K C C C K C K C C K
Lactose K K K K C C C K C K C C C
Melibiose C C C C C C K C C C C C C
D-Raffinose C C C C C C C C C C C C CSorbitol C C C C C C C C C C C C C
H2S production K K K K K K K K K K K K K
Gelatin hydrolysis K K K K K K K K K K K K K
Starch hydrolysis K K K K K K K K K K K K KLipid hydrolysis K K K K K K K K K K K K K
C, tested positive/utilized as substrate; K, tested negative/not utilized as substrate.
H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–19741972
Further, it has been reported that multiple strains of closely
related bacteria to dominate the rhizosphere soils of paddy
(Chin et al., 1999). The phylogenetic positions of the four
best performing strains: Enterobacter aerogenes (HK 201,
HK 34-1), Pantoea agglomerans (HK 14-1), Klebsiella sp.
(HK 34-2) is presented in Fig. 1. The sequences of three
representative strains were deposited in the GenBank
Table 4
Identification of PSB isolates by GC-FAME and 16S rDNA sequencing from rhi
Isolate GC-FAME identification Similarity (%)
HK 11-1 Enterobacter cancerogenus 64.9
HK 14-1 Enterobacter cloacae 82.7
HK 17-1 Enterobacter cloacae 82.9
HK 18-3 Kluyvera ascorbata 37.6
HK 20-1 Klebsiella pneumoniae 14.6
HK 23-2 Klebsiella pneumoniae 14.6
HK 24 Kluyvera ascorbata 60.6
HK 34-1 Kluyvera ascorbata 5.1
HK 34-2 Klebsiella pneumoniae 92.1
HK 52-1 Klebsiella planticola 83.1
HK 68-1 Enterobacter cancerogenus 7.6
HK 68-3 Klebsiella pneumoniae 43.8
HK 69 Klebsiella pneumoniae 89.1
nucleotide sequence data library under the following
accession numbers: AY335552 (HK 14-1, P. agglomerans),
AY335553 (HK 34-2, Klebsiella sp.) and AY335554
(HK 20-1, E. aerogenes).
Application of bacterial inoculants as biofertilizers has
been reported to result in improved plant growth and
increased yield (Bashan and Holguin, 1998; Vessey, 2003).
zosphere soil samples
16S rDNA identification Identity (%)
Pantoea sp. 98
Pantoea agglomerans 99
Pantoea sp. 96
Enterobacter cloacae 99
Enterobacter aerogenes 99
Klebsiella sp. 98
Enterobacter cloacae 99
Enterobacter sp. 95
Klebsiella sp. 98
Enterobacter cloacae 99
Enterobacter aerogenes 99
Klebsiella sp. 99
Klebsiella sp. 98
Fig. 1. Phylogenetic tree showing the relationships among the PSB isolates and between representatives of other related taxa. The tree was constructed by using
the MEGA2 after aligning the sequences with Megalign and generating evolutionary distance matrix inferred by the neighbor-joining method using Kimura
parameter 2. The numbers at the nodes indicate the levels of bootstrap support based on data for 1000 replicates; values inferred greater than 50% are only
presented. The scale bar indicates 0.005 substitutions per nucleotide position.
H. Chung et al. / Soil Biology & Biochemistry 37 (2005) 1970–1974 1973
Though no direct correlation could be established between
in vitro solubilization of P, plant P accumulation and
available soil P, the results of this study make these isolates
attractive as phosphate solubilizers. It requires further in-
depth studies based on the plant growth promoting activities
of these isolates under pot culture as well as field conditions
before they are recommended as biofertilizers.
Acknowledgements
The authors feel grateful to the Agriculture Research and
Promotion Center, Ministry of Agriculture and Forestry,
Korea for the support rendered by them. Sundaram Seshadri
also acknowledges the Korea Science and Engineering
Foundation for the financial assistance through Brain pool
fellowship.
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